An important problem in myocardial biology is how extracellular signals activate cardiac gene transcription. A gene activation method might provide a molecular switch to purposefully alter the cardiac phenotype in vivo. The applicant is studying the activation of cardiac myocyte growth and gene transcription by catecholamines acting through an a1-adrenergic receptor (AR). The focus has been on regulation of two contractile protein isogenes, the b-myosin heavy chain (MHC) and skeletal a-actin (skACT), since they exemplify a "fetal program", conserved in many models of hypertrophy in vivo and in culture. By studying the bMHC and skACT promoters in cultured cardiac myocytes, the applicant has identified a DNA sequence, enhancer core/M-CAT element, that is a response element for activation of these promoters by a1-AR stimulation. The applicant also has identified the transcription factor that interacts with this response element, transcriptional enhancer factor-1 (TEF-1). Although TEF-1 was cloned originally in the context of a viral promoter, gene knockout has shown that TEF-1 is indispensable for growth of the fetal heart in vivo. The applicant has cloned TEF-1 from rat cardiac myocytes. He finds seven TEF-1 isoforms, only a few of which had been recognized previously, probably produced by alternative splicing. By RNase protection assay, the mRNA for one isoform is expressed in adult cardiac and skeletal muscle, but not in brain or liver. When co- transfected with a b-MHC promoter, five of the TEF-1 isoforms activate, and two repress, and both actions are M-CAT site-dependent. Thus, cardiac myocytes are the first cell type in which transactivation by TEF-1 has been seen and heart may contain a muscle-specific TEF-1 isoform. Stimulation of myocytes with an a1-AR agonists translocates b-protein kinase C (PCK) to the nucleus, causes phosphorylation of TEF-1 and increases TEF-1 DNA binding and protein amount. b-PCK stimulates the M-CAT element and phosphorylates TEF-1 in vitro. Thus, these results suggest a model wherein stimulation of a a1-AR at the myocytes surface activates b-PKC and phosphorylates TEF-1. This activates TEF-1 and increases its DNA binding and/or transactivating functions. Activated TEF-1 then increases transcription of its target genes. Two hypotheses will be explored in this proposal. First, TEF-1 is phosphorylated by a1-AR stimulation through PKC and this activates its functions. Secondly, The TEF-1 phosphorylation mechanism can be used to alter the cardiac molecular phenotype independent of the receptor, in cultured myocytes, and in intact heart. To test these ideas, two questions will be asked. First, are there cardiac-specific TEF-1 isoforms, and do these differ in DNA binding, transactivation, and translation? Secondly, does b-PKC stimulation produce phosphorylation of specific residues on TEF-1? Are these the same residues phosphorylated by a1-AR? Is PCK unique in this respect? Finally, do alteration of TEF-1 activity change transcription in myocyte culture and in the intact animal?